MVST BOD & NST PART IB
Pathology Practical Class 12
Thurs. 15th & Fri. 16th Nov. 2012
Viral Haemagglutination/Histopathology
1. TITRATION OF VIRUSES BY HAEMAGGLUTINATION
Many viruses have surface proteins which are responsible for binding to the plasma membrane
of the host cell. As a result some viruses agglutinate cells of some species, and this
characteristic can be used to titrate virus preparations. The influenza viruses efficiently
agglutinate fowl red cells. Serial dilutions of virus are made, and equal volumes of a 1% fowl
red cell suspension are added to each virus dilution. The end point occurs when insufficient
virus is present to cause agglutination.
The source of influenza virus in this experiment is the embryonated hen’s egg. Many animal
viruses can be cultivated in the fertile egg at about 8-12 days incubation. Some, such as
influenza and parainfluenza viruses, grow well in the allantoic cells, and are released in large
amounts into the allantoic cavity. Others, such as herpes viruses and poxviruses, produce
discrete, countable lesions (pocks) on the chorioallantoic membrane. An approximate anatomy
of the fertile egg is shown in the accompanying General Notes.
The technical staff in Virology have taken eleven day old embryonated eggs, drilled a hole in the
shell to facilitate inoculation, and added 0.1 ml of a suspension of influenza A/PR/8/34 (PR8)
virus into the allantoic cavity of each egg. The hole was sealed with wax and the eggs incubated
blunt end up for 48 hours at 37˚C. The virus was recovered from the allantoic cavity and an
aliquot diluted 1/10 in PBS (phosphate-buffered saline), is provided for your experiment.
Materials
1.
One cupped Perspex tray - please do not write on these trays. Make a sketch of the tray
in your notes and annotate the sketch with your experimental layout, which you can then fill in
with your results.
2.
Suspension of virus ‘Flu’. This is a 1/10 dilution prepared as described above. Please
recap firmly after use.
3.
Blue or black capped 100 ml glass bottle containing 1% suspension of fowl red blood
cells (rbc) in PBS. Remember to gently but thoroughly swirl immediately before use so
that all the cells are in suspension. Again, please recap properly after use.
4.
Plastic 20 ml tube containing PBS.
5.
Pipettors and tips.
Method
1.
2.
3.
4.
5.
6.
7.
Work in pairs.
Do not write on the trays with marking pens.
Place 0.2 ml of PBS in each of 8 cups in the perspex tray. Make sure that the cups are
clean!
Add 0.2 ml of the virus to the first cup, and then make serial doubling dilutions up to
1:2560.
Place 0.2 ml PBS in one cup as a negative control.
Add 0.4 ml of the 1% suspension of red blood cells (swirl before adding) to each cup. Mix
by gentle shaking and leave at room temperature. Allow to settle without further
disturbance or mixing. You will need to use the computer for the next part of the
practical so rearrange your bench so you can do so without disturbing the
haemagglutination tray once you have added the rbc. Examine at 20 and at 40 minutes
and determine the haemagglutinating titre of the virus suspension (units are HA units
per ml where an HA unit is that which is sufficient to agglutinate 0.4ml of a 1%
suspension of rbc).
Each pair of students should do at least two titrations to check for accuracy.
N.B.: The results of this test are seen most clearly if the tray is left to stand on a piece of white
paper. It is important not to move the tray once the experiment has been set up.
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Catalogue Number
Small Image
A_IN_VI_BO_01.jpg
Titration of viruses
Image Map
Large Image
Titration of viruses
2. VIRUSES AND DISEASE
The multiplication of viruses may have one or more of five effects on the affected cell which can
be seen histopathologically.
1.
Necrosis. Virus multiplication usually kills the infected cell. Such cells undergo the usual
necrotic changes terminating in total disruption. Neurones affected by poliomyelitis virus are a
typical example of this effect. Cell death is sometimes preceded by other changes such as cell
fusion or loss of adhesion (e.g. herpes simplex virus or varicella zoster virus).
2
Proliferative changes followed by necrosis. Effect seen particularly with poxviruses,
where there is an initial and short-lived hyperplasia of the affected cells, followed by necrosis.
These changes occur in the typical 'pock', together with an extensive inflammatory reaction.
3.
Proliferative changes leading to neoplastic transformation. Occasionally virusinfected cells begin to multiply at a rapid rate. This change is produced by the so-called
'tumour' viruses, and the affected cells may or may not produce virus.
4.
Inclusion bodies. A characteristic feature of the multiplication of many types of virus is
the production of inclusion bodies. These structures may occur in the nucleus or the cytoplasm
of affected cells, and usually represent the sites of virus development. They have staining
characteristics which make them readily distinguishable from normal cellular elements.
Inclusion bodies are frequently so distinctive that their presence can be used as a diagnostic
sign of virus infection.
5.
Secondary infection. Damage to tissue by a virus, particularly epithelial tissue,
predisposes to secondary bacterial infection, i.e. opportunistic infection.
HISTOPATHOLOGY OF VIRUS DISEASES
The oil immersion lens is not required in viewing these slides:
12.1
Spinal cord: Acute Anterior Poliomyelitis : necrosis
55.245
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_SD_03.jpg
Spinal cord - Poliomyelitis
Spinal cord - Poliomyelitis
A_IN_VI_SD_04.jpg
Spinal cord - Poliomyelitis
Spinal cord - Poliomyelitis
Acute inflammation affecting both the cord and meninges. Blood
conspicuously dilated with characteristic perivascular cuffing of
Dead motor neurones of the anterior horns are replaced by
neutrophil polymorphs and macrophages. Compare with the normal
NE3
vessels are
leukocytes.
clusters of
section.
Spinal cord: normal
54.346
Catalogue Number
Small Image
N_NE_SD_01.jpg
Spinal cord - normal
Image Map
Large Image
Spinal cord - normal
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12.2
Skin: Herpes Zoster (shingles). Necrosis, cell fusion, nuclear changes,
loss of adhesion
71.150
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_SK_01.jpg
Skin – Herpes Zoster
Skin – Herpes Zoster
A_IN_VI_SK_02.jpg
Skin – Herpes Zoster
Skin – Herpes Zoster
A_IN_VI_SK_20.jpg
Skin – Herpes Zoster
Skin – Herpes Zoster
There are large numbers of infected epithelial cells surrounding fluid-filled
vesicles (blisters). Cell fusion results in appearance of multinucleate cells and
many cells have lost adhesion and float into the vesicle. Note the unusual
staining characteristics of the nucleus - a dense central core separated from
the nuclear membrane-which probably results from the breakdown of
chromatin that accompanies virus replication. This dense core is sometimes
called a "nuclear inclusion body".
12.3
Rabbit cornea: Vaccinia : inclusion bodies and hyperplasia
72.849
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_EY_01.jpg
Rabbit cornea - Vaccinia
Rabbit cornea - Vaccinia
A_IN_VI_EY_02.jpg
Rabbit cornea - Vaccinia
Rabbit cornea - Vaccinia
Low power examination of the cornea will reveal a thickened and perhaps
ulcerated area. The cells in this area contain characteristic paranuclear
eosinophilic inclusion bodies. Note the mitotic figures reflecting proliferation of
epidermal cells around the infectious focus in response to the virus-specific
epidermal growth factors.
12.4
Skin: Molluscum Contagiosum : inclusion bodies and hyperplasia
77.272
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_SK_03.jpg
Skin – Molluscum
contagiosum
Skin – Molluscum
contagiosum
A_IN_VI_SK_04.jpg
Skin – Molluscum
contagiosum
Skin – Molluscum
contagiosum
A_IN_VI_SK_05.jpg
Skin – Molluscum
contagiosum
Skin – Molluscum
contagiosum
A cup-shaped lesion of the epidermis, which is greatly thickened and in its
upper layers contains very large eosinophilic cytoplasmic inclusion bodies. Like
the previous example, this disease is caused by a pox virus, but the
histological changes are more dramatic.
12.5
Bovine cerebellum: Rabies: inclusion bodies
70.276 & 70.275
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_BR_03.jpg
Cerebellum - Rabies
Cerebellum - Rabies
A_IN_VI_BR_07.jpg
Cerebellum - Rabies
Cerebellum - Rabies
A_IN_VI_BR_08.jpg
Cerebellum - Rabies
Cerebellum - Rabies
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Some Purkinje cells contain cytoplasmic Negri inclusion bodies, which are
eosinophilic and about the size of a red blood cell.
12.6
Lung: Cytomegalovirus : inclusion bodies and cellular hypertrophy
71.959
Catalogue Number
Small Image
Image Map
Large Image
A_IN_VI_LU_01.jpg
Lung Cytomegalovirus
Lung Cytomegalovirus
A_IN_VI_LU_06.jpg
Lung Cytomegalovirus
Lung Cytomegalovirus
There are one or two small foci of inflammation where the alveolar epithelial
cells in these areas are enlarged and contain the typical 'owl eye' intranuclear
inclusion bodies. Cytomegalovirus is a member of the herpesviruses. Note the
similarity between the appearance of the nuclei in this slide and in the herpes
zoster slide.
3. DATA HANDLING EXERCISE FROM LAST TIME
Discuss this with your demonstrator; there will be opportunity to do this next time as well. If
you do not have your notes for class 11 with you, make a note to bring them to class 13 along
with the accompanying general notes which you will need in order to benefit fully from practical
13.
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